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The Evaluation of Essential Oils for Antimicrobial Activity

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The Evaluation of Essential Oils for Antimicrobial Activity Grand Valley State University ScholarWorks@GVSU Student Summer Scholars Undergraduate Research and Creative Practice Summer 2014 The vE aluation of Essential Oils for Antimicrobial Activity Dallas Rohraff Grand Valley State University Roderick Morgan Grand Valley State University Follow this and additional works at: http://scholarworks.gvsu.edu/sss Recommended Citation Rohraff, Dallas and Morgan, Roderick, "The vE aluation of Essential Oils for Antimicrobial Activity" (2014). Student Summer Scholars. 124. http://scholarworks.gvsu.edu/sss/124 This Open Access is brought to you for free and open access by the Undergraduate Research and Creative Practice at ScholarWorks@GVSU. It has been accepted for inclusion in Student Summer Scholars by an authorized administrator of ScholarWorks@GVSU. For more information, please contact [email protected]. The Evaluation of Essential Oils for Antimicrobial Activity Abstract The emergence of antibiotic resistant bacteria is of pressing concern as health care associated infections kill 99,000 people a year in the U.S. alone. Researchers are currently looking for new antibiotics in alternative sources. Essential oils are traditionally known to have medical benefits, and cinnamon, tea tree, and eucalyptus oils have shown antibiotic activity. Initial testing via standard microbiological protocols found minimum inhibitory concentration (MIC) values of 0.039% for cinnamon, 1.25% for tea tree, and 0.313% for eucalyptus. All three oils proved effective against both Gram-positive and Gram-negative bacteria. Cinnamon bark oil, Cinnamomum cassia Blume, appeared most effective. More thorough microbiological analysis revealed it to be bactericidal and retained antibacterial activity in the presence of human serum protein. The results revealed cinnamon bark oil may contain a promising novel antibiotic. Introduction Infectious diseases, particularly those caused by bacterial microorganisms, are still among the top causes of mortality in the world. The rate of infections caused by bacteria that have acquired antibiotic resistance is a staggering proportion. This is especially concerning in hospital settings where 1.7 million health care-associated infections are acquired and kill 99,000 people every year. It is estimated that these infections cause between $28 and $32 billion dollars to the health care industry (5). Several resistant strains, such as multi-antibiotic resistant Staphylococcus aureus (MRSA), carbapenem resistant enterobacteriaceae (CRE), Vancomycin Resistant Enterococci (VRE), and antibiotic resistant hypervariant Clostridium difficile (Cdiff), are commonly acquired through a nosocomial infection and demonstrate the necessity of novel antibiotics to combat bacteria that have become resistant to currently used antibiotics. Bacteria can become resistant to antibiotics in a number of ways, one of which is random mutation. Mutations can occur in DNA which would usually code for antibiotic sensitivity and in turn code for resistance (9). Once this occurs, the bacterium can spread the mutation when it produces daughter cells (9). This mutation occurs in about one per million to one per billion cells (9). Another mode by which bacteria can become resistant is by the transfer of plasmids that code for resistance from one cell to another through conjugation. For either circumstance, once one bacterium becomes resistant, the number of resistant bacteria will begin to increase (9). There has been much concern in recent years over the misuse and overuse of antibiotics. Misuse can occur with the prescription of antibiotics when they are not necessary, and some worry antibiotics are being overused in the agriculture and livestock business where recommended doses are commonly added to feed for health purposes and increased rates of livestock growth (9). When this occurs, resistant bacteria survive and are artificially selected for and allowed to replicate. This can lead to rapid growth of resistant bacteria which are resistant to current antibiotics. There are few treatments currently available that can combat these antibiotic resistant bacteria, and the necessity of novel antibiotics is becoming evident. Unfortunately, progress in the development of new antibiotics has tapered off as several large pharmaceutical companies have decreased their infectious disease discovery programs. Much of the current research is looking toward synthesizing novel derivatives which are modeled after current antibiotics (11). There has been low success rates in finding novel antibiotics with these derivatives, and this could be because a majority of the antibiotics used today are produced naturally from soil streptomycetes and fungi (11). While many large companies are focusing on manmade products, and finding little success, some researchers are once again looking toward natural sources for new and improved antibiotics (11). One natural product, which has been around for thousands of years, may hold the key to finding new antibiotics: essential oils derived from plants. EOs are aromatic liquids generally made through a steam distillation process of plant material which can be traced back over 2000 years to Egyptian, Persian and Indian roots; but can also be made through extraction, enfleurage, fermentation and expression (2). EOs are typically made from aromatic plants of warm, tropical regions and can be made from nearly any plant organ, such as leaves, bark, herbs, roots, seeds, stems, and fruits. Climate, soil, plant organ, age of plant, and harvest time all affect the quality and quantity of an essential oil yield (1). EOs have been used for pharmaceutical purposes since the 13th century in some areas of Europe (7), but their use was not widespread until they were traded in London in the 16th century (2). French physician, Du Chesne, noted that by the 17th century, EOs were a common medical practice in Europe, with pharmacies stocking 15 to 20 different essential oils at a time (7). It was not until 1881 that EOs were tested for antimicrobial properties, when De La Croix examined vapors (2). By the 19th and 20th centuries, EOs were increasingly used for fragrances and flavoring different foods (7). Today, only a small percentage of the essential oils created are used for aromatherapy. EOs have countless uses, such as perfumes, cosmetics, dentistry, food preservatives and flavoring, and more (1). In recent years, more research has been done to determine the antimicrobial nature of numerous EOs against different bacteria (1, 7). Essential oils have also been found to have antiviral, antiparasitic, insecticidal, antitoxigenic, antiseptic, and tumor inhibiting activities as well (1, 2, 10). Because EOs are made from various plant components, it stands to reason that they would contain properties that the plant uses to protect itself from bacteria, viruses, and unwanted insects, while containing fragrances that could help attract insects that would be beneficial for pollination (1). Studies show EOs have the potential to solve a variety of different medical problems, including the crisis of antibiotic resistance of infectious bacteria. We initially tested several essential oils using the Agar Disk Diffusion Test, or the Zone of Inhibition Test, to determine if the oil had any antibiotic activity. If an oil demonstrated a promising zone, further testing for Minimum Inhibitory Concentrations (MICs) were performed against Staphylococcus aureus and Escherichia coli. Cinnamon bark oil showed a very low MIC, meaning that it remained effective in small doses, and was tested using a Time Kill experiment to determine if it is bacteriostatic or bactericidal. Methods Essential Oils The essential oils tested were from a variety of different brands. The following oils were Plant Therapy brand: pine (Pinus Sylvestris), cinnamon bark (Cinnamomum cassia Blume), spearmint (Mentha spicata), peppermint (Mentha piperita), and juniper berry (Juniperus communis). Lavender (Lavandula officinalis) and ginger (Zingiber officianale) were Now Essential Oils. Orange oil was of the brand LorAnn Oils. The tea tree (Melaleuca alternifela), eucalyptus oils tested were Sundown Naturals and Aura Cacia, respectively. Anthranilic Acid Derivative- GV-2 GV-2 was prepared by the Chemistry Department at GVSU and identified using analytical (C, H, N) and spectral (IR, HNMR, CNMR, Mass) data. GV-2 shows antimicrobial activity against Gram-positive bacteria, but is not effective against those that are Gram-negative. GV-2 is used as a positive control against Staphylococcus aureus and a negative control against Escherichia coli for this reason. GV-2 has an MIC value of 16.0 µg/mL against S. aureus, but this value increases in the presence of Human Serum Protein to 128 µg/mL. Zone Of Inhibition Test Initial testing to determine antimicrobial activity of the test compounds was by the zone of inhibition test, or agar disc diffusion method (4). The zone of inhibition test, begins by swabbing a plate with overnight cultures of Staphylococcus aureus and Escherichia coli to create a “lawn.” Five microliters of essential oil were pipetted onto 6 millimeter sterile paper disks which were placed atop the bacterial “lawn” and incubated for 18-24 hours at 37oC. Areas of clearing, or zones of inhibition, around the disks after incubation are measured and indicate that the compound has some antimicrobial activity. Diameter of zones of inhibition were measured in millimeters and recorded. Determination of MIC The MIC’s of the tested essential oils were determined by the Clinical
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